Human cells sense the earliest steps of infection by HIV and respond almost immediately. We propose to investigate the cellular sensing apparatus, the intermediate signaling pathways, and the effector systems mobilized in response to the early steps of HIV infection. The cellular response has evolved in part to help contain infection, for example by induction of the interferon system. Pretreatment of cells with interferon causes a change in transcriptional program, resulting in induction of host systems that drastically reduce accumulation of HIV eDNA. Another host response to infection seen in some cells involves recognition of the HIV eDNA as DNA damage, with induction of a cellular DNA damage response and consequent apoptosis of a portion of the infected cells. We will investigate the mechanism by which cells sense early viral infection and respond by inhibiting viral replication. We will use a series of recently developed methods for this study, including RNAi, fluorescence-monitored PCR, and transcriptional profiling. One highlight of our preliminary data is the first demonstration that HIV replication can be blocked specifically and efficiently by RNAi. In the parent grant, "Mechanism of HIV DNA integration," we are studying the structure and function of HIV replication intermediates ("preintegration complexes," PICs) formed during the early steps of infection. Here we propose to expand the study to investigate the host responses to the presence of these replication complexes in cells. This project is exploratory in nature, since we do not know in advance the pathways we will find, and the main techniques involved are newly developed. We expect that the study will involve considerable methods development, particularly in bioinformatics and large-scale applications of RNAi. We propose to carry out the following Specific Aims: 1. Investigate RNAi in mammalian cells as 1) a tool to probe the host cell response to infection, and 2) an antiviral system in its own right. 2. Investigate the mechanism by which interferon treatment of target cells abrogates the early steps of HIV infection. 3. Use microarray technology to investigate the host cell response to early steps of infection.